Biological fuel cells, which use a biomass resource, have recently been proposed as being the next generation of energy, due to the high energy efficiency and low environmental impact thereof. Organisms, including microorganisms, generate ATP and other chemical energy substances (bonding energy) in in vivo metabolic processes involving the oxidative decomposition of carbohydrates, proteins, lipids, and the like by enzymes and other biological catalysts, thus acquiring the energy needed for life activity. A biological fuel cell is a power generation device that removes energy generated in such in vivo metabolic processes to an electrode as electrical energy. In particular, enzyme fuel cells, which conjugate the electrode reaction and a substrate-specific catalytic reaction of an enzyme, have been attracting attention as clean power sources that are especially safe and have an especially low environmental impact, because enzyme fuel cells are able to use, as fuel, compounds present in the environment, such as sugars and amines.
The selection of an enzyme that will serve as an electrode catalyst is a very important element in the construction of an enzyme fuel cell. For the anode (negative electrode) side, an enzyme that oxidatively decomposes the fuel will be selected, while an enzyme that reduces oxygen will be selected for the cathode (positive electrode) side. For example, glucose dehydrogenase is used as the anode-side catalyst in a case where, for example, glucose is to serve as the fuel. By contrast, a laccase or the like could be used as the cathode-side catalyst (Patent Document 1). Laccases are enzymes that are known to be widely present in microorganisms, fungi, plants, and so forth. For example, CotA laccase from Bacillus subtilis and other laccases have been reported, the sequences thereof determined and the crystal structures thereof analyzed (Non-patent Document 1, Non-patent Document 2).
Successfully putting an enzyme fuel cell to practical use hinges on the successful construction of an electrode having an enzyme immobilized thereon, and thus enzyme electrodes whereby the catalytic functions of enzymes can be maximized are being constructed. A variety of electrodes where the electrode surface has, immobilized thereon, either an enzyme or an electron-transfer mediator that mediates the electron transfer between the electrode and the enzyme have been reported to date. Reported examples include an enzyme electrode where direct bonding between a hydrophobic group of a membrane-bound enzyme and a hydrophobic group of a carbon base material has immobilized the membrane-bound enzyme onto the carbon base material (Patent Document 2), as well as an enzyme electrode where an enzyme configured to be a protein that includes a cytochrome complex (cytochrome C) site has been immobilized onto an electroconductive base material made of carbon having a hydrophobic surface (Patent Document 3). It has also been reported that in the process of immobilizing an enzyme or an electron-transfer mediator onto an electroconductive base material, an electrode having enzymes immobilized thereon exhibiting excellent electrode performance can be manufactured by bonding the mediator to an organic polymer chain to form a polymer-mediator composite, dispersing same in a solvent having an electric permittivity of 24 or less to improve dispersibility, and coating the surface of the electroconductive base material with the resulting dispersion (Patent Document 4), and further that adding an organic solvent to the solution used in the process of immobilizing the enzyme onto the electroconductive base material makes it possible to readily promote osmosis of the enzyme into the interior of the electrode and construct an electrode where the enzyme has been immobilized on the surface of the interior of the electrode in a three-dimensional and high density fashion, without adversely affecting the activity of the enzyme, even though the electrode may have a very complex structure (Patent Document 5). In another report, electrodes having a structure where a positive electrode and a negative electrode face each other with an electrolyte interposed therebetween were constructed, among which, as one example of the electrode, an electroconductive base material made of carbon felt was used, the enzyme being immobilized on the electrode by soaking the electroconductive base material with the enzyme (Patent Document 6).
However, in the prior art described above, all instances involved using an enzyme solution in a solution state where the enzyme has been dispersed into water or a buffer solution to immobilize the enzyme onto the carbon base material or other electroconductive base material. For this reason, a problem has emerged in that the enzyme on the electrode has become unstable and an electric current value that is consistent with the amount of enzyme binding cannot be obtained. Another problem has emerged in that the types of enzyme that can be applied have been limited, the applications of the electrode disclosed in Patent Document 2 being limited to membrane-bound enzymes and the electrode disclosed in Patent Document 3 not being applicable to enzymes having a hydrophilic surface, and so forth.
In order to obtain an adequate electric current value in an enzyme electrode, there must be smooth electron transfer between the enzyme and the electrode via the electron-transfer mediator. For this reason, it has been necessary to have a greater amount of enzyme present at a location in close proximity to the surface of the electrode. This signifies that the electrode performance of the enzyme electrode is significantly affected by the relative positional relationship between the enzyme, the electron-transfer mediator, and the electrode. For this reason, raising the concentration in the process of immobilizing the enzyme onto the surface of the electrode has been regarded as very important in order to obtain an enzyme electrode that exhibits excellent electrode performance.
There are limitations, however, to the use of a highly concentrated solution in a case where the enzyme is to be immobilized onto the electroconductive base material in a solution state. An enzyme is constituted of amino acids that are either hydrophilic or hydrophobic, and, when in a solution, adopts such a structure that the hydrophilic amino acids are present on the surface. For this reason, precipitation will generally not take place at concentrations of about several milligrams/milliliters. However, when the enzyme is highly concentrated, there are incidental interactions between the hydrophobic portions when the enzyme molecules come into close proximity to each other; aggregation and precipitation take place as a result, leading to denaturing of the enzyme and exposure of the enzyme in an unstable state. In addition, once precipitation takes place, the precipitation will continue to increase at an accelerated pace. Therefore, when the enzyme concentration in an enzyme solution is increased (generally, when the concentration is in excess of 50 mg/cm3), the dispersibility of the enzyme in the solution is worsened, and the enzyme will be immobilized onto the surface of the electroconductive base material in an aggregated, i.e., denatured state. This causes electron transfer on the electrode to no longer proceed in a smooth manner, and a problem emerges in that the resulting enzyme electrode will have poor electrode performance.
As an example, according to a specific disclosure made in Patent Document 2, the enzyme concentration in a solution used in the process of constructing an electrode is 0.57 mg/mL, i.e., the maximum amount of membrane-bound enzyme immobilized onto the carbon base material is 1.11 μg/cm2. According to a specific disclosure made in Patent Document 3, it is stated that a glassy carbon electrode is constructed by adding 5 μL of a 1 mg/mL enzyme solution in a dropwise manner thereon. Thus, since the enzyme concentration in the solution used is 1 mg/mL, the maximum amount of membrane-bound enzyme immobilized onto the carbon base material is 71.4 μg/cm2. Further, according to a specific disclosure made in Patent Document 4, it is stated that an electrode is constructed by adding 8 μL of a phosphate buffer solution having an enzyme concentration of 5 mg/mL in a dropwise manner onto a carbon paper surface. Thus, the enzyme concentration in the solution used is 5 mg/mL; calculated on the basis thereof, the maximum amount of enzyme immobilized onto the carbon sheet is 51.0 μg/cm2. According to a specific disclosure made in Patent Document 5, 13.8 mg of an enzyme is dissolved in 200 μL of a buffer solution and the enzyme solution is added in a dropwise manner onto a glassy carbon disc electrode surface; therefore, the enzyme concentration in the solution used is 69 mg/mL. According to a specific disclosure made in Patent Document 6, the enzyme concentration in the solution used is 50 mg/mL. In the prior art, thus, it has been presumed that the enzyme concentration used in immobilization is as described above.
Factors whereby an amount of electric current that is consistent with the amount of enzyme binding might not be obtained also include the fact that there is inadequate optimization of the directionality (orientation) of the enzyme binding on the electrode. When in a solution, the enzyme is present in a disordered state, without a unified orientation. When immobilization is carried out in a solution state, therefore, the enzyme binds to the electroconductive base material with a random directionality, and this leads to a decrease in output and the like. For this reason, the ability to control the orientation of the enzyme on the electrode has been a technical problem requiring improvement, from the point of view of electrode performance. Furthermore, in immobilization in a solution state, immobilizing the enzyme onto the electrode while a constant dispersibility is upheld is difficult, and for this reason a problem has emerged in that the enzyme readily dissociates from the electrode within the electrolyte solution, and there is a decline in the electric current value when the oxidation current is measured.